Abstract

This study was to evaluate the effect of sweet or bitter cassava foliage, rice distillers’ byproduct and biochar on methane production in an in vitro incubation system with ensiled cassava root as the energy source. The design was a 2*2*2 factorial with 4 replications: rice
distillers’ byproduct (RDB) at 0 or 4% of substrate DM, biochar: 0 or 1% of substrate DM and leaves of sweet or bitter cassava at 30% of substrate
DM. Urea (3% of ensiled root DM) and sulphur-rich minerals (2% of substrate DM) were included in the fermentation medium. The total substrates equivalent
to 12g DM were put in the incubation bottle, followed by 960 ml of buffer solution and 240 ml of rumen fluid “obtained from a cattle immediately
after being slaughtered”. The bottles were then filled with carbon dioxide and incubated at 38 0C in a water bath for 24 hours.

Methane production was reduced when: leaf meal from bitter rather than sweet cassava was the protein source in an in vitro fermentation of ensiled cassava
root; biochar was added to the fermentation medium.

Introduction

Greenhouse gases (GHG) will have to peak by 2020 and drop by 75-80 per cent
in the period to 2050 to limit global warming to two degrees (The Climate
Group 2008). The total GHG emissions in 2010 were estimated to have
increased by more than 6 per cent, and for 2011 were estimated to have
increased by 3.2 per cent (The Guardian 2011; IEA 2011). The methane
emissions from enteric fermentation in herbivorous animals, especially
ruminants, are considered a major source of greenhouse gases (Stavi and Lal
2013).

Cassava (Manihot esculenta Crantz) is grown in over 90 countries
and is a most important food crop worldwide. It is the primary staple for
more than 800 million people in the world (Lebot 2009). Of importance in a
warming world is that it appears that cassava is potentially highly
resilient to future climatic changes and according to Jarvis et al (2012)
“could provide Africa with options for adaptation whilst other major food
staples face challenges”.

Cassava foliage is considered to be a good source of bypass protein for
ruminants (Ffoulkes and Preston 1978; Wanapat 2001; Keo Sath et al 2008). It
has been fed as a major component of the diet for sheep (Hue et al 2008),
goats (Ho Quang Do et al 2002; Dung et al 2005; Phengvichith and Ledin 2007;
Seng Sokerya and Preston 2003) and cattle (Wanapat et al 2000; Thang et al
2010) in fresh, wilted or dried form.

Cassava leaves are known to contain variable levels of condensed tannins; about
3% in DM according to Netpana et al (2001) and Bui Phan Thu Hang
and Ledin (2005). Condensed tannins at moderate levels are known to have
positive effects on the nutritive value of the feed by forming insoluble
complexes with dietary protein, resulting in "escape" of the protein from the
rumen fermentation (Barry and McNabb 1999). Numerous studies have also shown the
potential of the tannin content in cassava leaves to play an anthelminthic role
for the control of nematode parasites in ruminants (Seng Sokerya and Preston
2003; Seng Sokerya et al 2009; Netpana et al 2001; Khoung and Khang 2005).
Condensed tannins (CT) are also reported to decrease methane production and
increase the efficiency of microbial protein synthesis (Makkar et al 1995;
Grainger et al 2009). Reductions of CH4 production due to presence of
tannins were reported by Carulla et al (2005), Waghorn et al (2002), Grainger et
al (2009) and Woodward et al (2004), apparently through a direct toxic effect on
methanogens.

Previous research showed that methane production in a rumen in vitro
fermentation system was reduced when the protein source was leaf meal
derived from “bitter” as opposed to “sweet” varieties of cassava (Le Thuy
Binh Phuong et al 2011).

Rice distillers’ by-product is another potential source of high quality
protein in rural areas of Lao PDR. Rice distillers’ by-product is the
residue after distilling the alcohol derived by yeast fermentation of sticky
rice (Taysayavong Lotchana and Preston 2010). The farmers traditionally use
it as a mixture with other feeds such as rice bran and broken rice in diets
for fattening pigs (Oosterwijk et al 2003). The farmers in Vietnam also use
rice distillers’ by-product (known as “hem”) as a traditional feed for pigs
(Luu Huu Manh 2000). The protein content of "hem" ranged from 17 to 33%
(mean of 23%) in dry matter with a well-balanced array of amino acids (Luu
Huu Manh et al 2003). These authors reported that this product could replace
completely the fish meal in growing and fattening pig diets with no loss of
performance.

Biochar derived from partial combustion of rice husks in an updraft gasifier
stove (Olivier 2010) reduced methane production in an in vitro
rumen incubation of cassava root meal and cassava leaf meal supplemented
with urea or potassium nitrate as the major fermentable N source (Leng et al
2012).

The objectives of the present study were to evaluate the effect of sweet or
bitter cassava foliage, rice distillers’ byproduct and biochar on methane
production in an in vitro incubation system with ensiled cassava
root as the energy source.

Materials and methods

Location and duration

The experiment was conducted in the laboratory of the Faculty of Agriculture
and Forest Resource, Souphanouvong University, LuangPrabang Province, Lao
PDR, from January to February, 2016

Treatments and experimental design

The design was arranged as a 2*2*2 factorial with 4 replications. The
factors were:

The basal substrate was ensiled cassava root and urea at 3% of the DM of the
ensiled root plus sulphur-rich minerals at 2% of substrate DM.

The in vitro system

The in vitro system was made from recycled “PEP” water bottles as
described by Inthapanya et al (2011) (Photos 1, 2 and 3).

Photo 1. The in vitro system

Photo 2. Measurement of methane production in the gas

Photo 3. The substrate residue filtered through cloth

Experimental procedure

The cassava root was chopped into small pieces around 1-2 cm of length, then
ensiled in sealed plastic bags for 7days. Cassava leaves (harvested from a
sweet and a bitter variety) were chopped into small pieces around 1-2 cm of
length, then dried in sunlight before grinding (1mm sieve).

The biochar was produced by burning rice husks in a top lit updraft (TLUD)
gasifier stove (Olivier 2010) at a temperature of 900-1000oC. The
biochar was ground through a 1 mm sieve. Rice distillers’ byproduct was
bought from farmers who produce “rice wine”.

Amounts of the substrates equivalent to 12g DM were put in the incubation
bottle in the in vitro system followed by 0.96 liters of buffer
solution (Table 1) and 240 ml of rumen fluid obtained from a steer
immediately after being slaughtered. The bottles was then filled with carbon
dioxide and incubated at 38 0C in a water bath for 24 h.

Table 1.
Ingredients of the buffer solution

Ingredients

CaCl2

NaHPO4.12H2O

NaCl

KCl

MgSO4.7H2O

NaHCO3

Cysteine

(g/liter)

0.04

9.30

0.47

0.57

0.12

9.80

0.25

Source: Tilly and Terry (1963).

Data collection and measurements

During the incubation the gas volume was recorded at 12 and 24h (Photo 1).
After each time interval, samples of gas were taken for measurement of the
methane concentration using an infra-red meter (Crowcon Instruments Ltd, UK)
(Photo 2). At the end of the incubation, the residual DM in the incubation
bottle was measured to determine mineralization of the DM (Photo 3).

Chemical analyses

The samples of ensiled cassava root, rice distillers’ byproduct, and cassava
leaf meals were analyzed for DM, ash and crude protein according to AOAC
(1990) methods.

Statistical analysis

The data from the experiment were analyzed by the general linear model
option of the ANOVA program in the Minitab software (version 16.0). The
sources of variation were: replicates, RDB, biochar, sweet or bitter leaves
of cassava, interaction RDB*biochar, RDB*cassava leaf, RDB*biochar*cassava
leaf and error.

Results

Chemical composition

The composition of the substrate ingredients is in Table 2.

Table 2.
Chemicals composition of feed in % DM

Items

DM

OM

CP

Ensiled cassava root

31.7

86.9

2.7

Rice-wine distillers’ BP

8.0

98.2

25.8

Cassava leaf meal

Sweet

88.7

94.1

22.5

Bitter

89.9

94.1

22.2

Biochar

78.9

13.3

-

Gas production

Gas production was not affected by rice distillers’ byproduct nor by biochar
but was less for bitter compared with sweet cassava leaf meal (Table 3).

Methane

The methane concentration in the gas increased with fermentation time (Table
3). Per unit of substrate methane production was not affected by RDB (Figure
1), was lower for bitter compared with sweet cassava leaves (Figures 2 and
6) and for addition of biochar (Figures 3 and 5). Substrate DM mineralized
was increased by RDB, and decreased by bitter compared with sweet cassava
leaves and by addition of biochar.

Table 3.
Mean values for gas production, percent methane in the gas, Digestibility and methane per unit of DM substrate

By-products

p

Cassava leaf

p

Biochar

p

SEM

RD0

RD4

Bitter

Sweet

BC

NBC

Gas production (ml)

0-12h

1109

1194

0.052

1103

1200

0.028

1119

1184

0.125

29.2

12-24h

741

716

0.537

684

772

0.038

703

753

0.223

28.2

Methane in the gas (%)

0-12h

21.5

22.7

0.002

21.4

22.8

0.001

21.0

23.2

<0.001

0.249

12-24h

25.3

26.1

0.046

24.8

26.6

<0.001

24.6

26.7

<0.001

0.274

Total gas, ml

1850

1909

0.42

1788

1972

0.018

1822

1938

0.123

51.1

Digested (%)

68.2

71.2

<0.001

66.8

72.6

<0.001

68.5

70.9

<0.001

0.407

CH4 (ml/ g DM substrate)

54.8

56.5

0.463

53.6

57.8

0.077

52.4

58.9

0.009

1.63

Figure 1. Effect of rice distiller’s by product with bitter or
sweet cassava leaf meal on methane per unit substrate

Figure 2. Effect of bitter or sweet cassava leaf meal with or
without rice distiller’s by product on methane per unit
substrate

Figure 3. Effect of biochar with or without rice distiller’s by
product on methane per unit substrate

Figure 4. Effect of rice distiller’s by product with
or without biochar on methane per unit substrate

Figure 6.
Effect of bitter or sweet cassava leaf meal with or
without biochar on methane per unit substrate

Discussion

The reduction in methane when leaf meal from bitter rather than sweet
cassava was the protein source is in agreement with the findings of Binh
Phuong et al (2011) in an in vitro rumen system. A similar result
was reported by Binh Phuong et al (2016, personal communication) in cattle
fed foliage from a bitter compared with a sweet cassava variety as a
supplement to ensiled cassava root pulp.

In research reported by Phuong et al (2012), there were only minor differences in the solubility of the protein between
bitter (31.9% soluble protein) and sweet (28.8 – 30.4% soluble protein)
cassava varieties. The higher
concentrations of cyanogenic glucosides (precursors of HCN) in leaves from
bitter compared with sweet cassava is a more likely explanation of the
reduced production of methane from leaves of bitter cassava. The research of
Eikmanns and Thauer (1984) and Smith et al (1985) supports the concept that
cyanide is somewhat toxic to methanogens or reduces their potential growth
by lowering the availability of sulphur by formation of thiocyanates (Majak
and Cheng 1984). Additions of 5, 10, and 25 mg 1itre-l cyanide
(from KCN or linamarin) temporarily inhibited methanogenesis in biodigesters
charged with cassava root waste (Cuzin and Labat 1992). The biodigester
methanogenic microflora were sensitive to the added cyanide.

Conclusions

Methane production was reduced when: leaf meal from bitter rather than
sweet cassava was the protein source in an in vitro fermentation of
ensiled cassava root; biochar was added to the fermentation medium

Acknowledgements

The authors acknowledge support for this research from the MEKARN II project
financed by Sida. Special thanks are given to Mr Sangkhom Inthapaya who
provided valuable help in the laboratory and I also acknowledge
Souphanouvong University, Faculty of Agriculture and Forest Resources,
Department of Animal Science, providing the facilities to carry out this
research.

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